Enzymes with aminopeptidase activity

ABSTRACT

The present invention relates to a enzyme exhibiting aminopeptidase activity, a method for producting said enzyme, and enzyme preparation containing said enzyme exhibithing aminopeptidase activity, and use of said enzyme for various industrial purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK96/00104 filed Mar. 15, 1996which claims priority under 35 U.S.C. 119 of Danish application 0262/95filed Mar. 16, 1995, respectively, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an enzyme exhibiting aminopeptidaseactivity, a method for producing said enzyme, an enzyme preparationcontaining said enzyme exhibiting aminopeptidase activity, and use ofsaid enzyme for various industrial purposes.

BACKGROUND OF THE INVENTION

Protein hydrolysates are being used in numerous food products.Traditionally protein hydrolysates were produced by acid hydrolysis, buttoday enzymatic hydrolysis is regarded as an attractive alternative.

One of the main problems of protein hydrolysates is that they oftentaste bitter. When using e.g. soy protein or casein, which are rich inhydrophobic L-amino acids, as the protein source, the proteinhydrolysate tends to have bitter taste. In general it is believed thatwhether the taste of proteins is bitter or not depends on the averagehydrophobicity of the L-amino acid residues, such as valine, leucine,isoleucine, phenylalanine, tyrosine and tryptophan.

A vast number of enzymes exhibiting peptidase activity are capable ofperforming enzymatic hydrolysis on vegetable, yeast and/or animalproteins, resulting in highly nutritious protein hydrolysates useful asfood additives in products such as soups, sauces, gravies, paste, tofu,bouillon, seasonings, baby formulas, snacks, ready-to-eat meals etc.

Peptidases

All peptidases or proteases are hydrolases which act on proteins or itspartial hydrolysate to decompose the peptide bond.

EP 427,385 (The Japanese R & D Association) discloses a genomic geneencoding an alkaline protease derived from yellow molds such asAspergillus oryzae.

JP-0-2002374 and JP-0-2002375 (Shokuhin), describes an alkaline proteasederived from Aspergillus oryzae for use in medicine, food, anddetergents.

SU-891777 (Khark) concerns a microbial protease from Aspergillus oryzae,which can be used in food, medicine etc.

JP-5-4035283 (Ajinomoto KK) discloses preparation of enzymes from e.g.Aspergillus oryzae exhibiting endopeptidase activity, which canhydrolyse proteins almost completely.

WO 94/25580 (Novo Nordisk A/S) describes a method for hydrolysingvegetable or animal protein by incubating with a proteolytic enzymepreparation derived from a strain of Aspergillus oryzae.

Aminopeptidases

A subgroup of peptidases (proteases) are called aminopeptidases and areclassified under the Enzyme Classification number E.C. 3.4.11(aminopeptidases) in accordance with the Recommendations (1992) of theInternational Union of Biochemistry and Molecular Biology (IUBMB)).

Aminopeptidases are capable of removing one or more amino terminalresidues from polypeptides.

JP-7-5034631 (Noda) discloses a leucine aminopeptidase derived fromyellow koji mold, which includes Aspergillus oryzae.

JP-7-4021798 (Zaidan Hojin Noda Sangyo) describes the production of misoby adding of a leucine aminopeptidase II prepared by cultivating anumber of molds, including Aspergillus oryzae strain 460 and strain IAM2616.

Van Heeke et al., Bioch. Biophys. Acta, (1992), 1131, 337-340, havedisclosed the cloning of a 30 kDa aminopeptidase from the bacteriaVibrio proteolyticus deposited at the American Type Culture Collectionunder the ATCC No. 15338.

Aspergillus oryzae 460 is known to produce a number of leucineaminopeptidases. The molecular weight of three of these was calculatedto 26,500, 56,000 and 61,000, respectively determined by gel filtration(Nakada et al., Agr. Biol. Chem, (1972), 37(4), 757-765; Nakada et al.,Agr. Biol. Chem, (1972), 37(4), 767-774; Nakada et al., Agr. Biol. Chem,(1972), 37(4), 775-782). The Aspergillus oryzae 460 strain is depositedat the American Type Culture Collection as A. oryzae (ATTC no. 20386).

Reduction of Bitter Taste of Protein Hydrolysates

EP 65,663 and EP 325,986 (Miles Inc.) concerns enzymatic hydrolysis ofproteins using a mixture of enzymes containing Aspergillus oryzaederived proteases. The obtained protein hydrolysate has a bland,non-bitter taste.

JP-4-7029577 (Asahi Electro-chemical Co.) concerns a protease, derivedfrom Aspergillus oryzae, which does not produce any bitter componentwhen decomposing protein.

Prior art discloses a plethora of enzymes exhibiting peptidase,aminopeptidase and other enzyme activities. Said enzymes may be derivedfrom a number of microorganisms, including the fungus speciesAspergillus oryzae.

In general products, useful for producing protein hydrolysates without abitter taste, comprise a mixture of peptidase and aminopeptidaseactivities.

It would therefore be desirable to be able to provide a single-componentenzyme (i.e. substantially without any side activity) exhibiting only anactivity useful for reducing the bitterness of protein hydrolysates usedin food products.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the result of SDS-PAGE analysis of supernatant from theAspergillus oryzae A01568 35 kDa aminopeptidase producing transformant.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a single-componentenzyme exhibiting an activity, which is particularly useful forpreparing improving bread products and for producing proteins and/orprotein hydrolysates without bitter taste for foodstuff.

The present inventors have surprisingly succeeded in isolating a DNAsequence encoding an enzyme exhibiting aminopeptidase activity, whichadvantageously may be used for improving the flavour, crust colour,crumb structure and dough stickiness of baked products. Further, saidnovel enzyme is useful for producing protein or protein hydrolysateswithout bitter taste.

The complete DNA sequence encoding said aminopeptidase makes it possibleto prepare single-component aminopeptidases.

The complete DNA sequence, shown in SEQ ID no. 1, encoding theaminopeptidase of the invention has, comprised in a plasmid, beentransformed into the bacteria strain Escherichia coli DSM no. 9965. Thiswill be described further below.

By a database alignment search it was found that the DNA sequence shownin SEQ ID No. 1 is novel. The highest degree of similarity and identitywas found to be 53% and 32%, respectively, to the above mentioned 30 kDaaminopeptidase from the bacteria Vibrio proteolyticus (ATCC No. 15338).

The inventors have characterized the precursor-form of theaminopeptidase consisting of a secretion signal and the 35 kDaaminopeptidase. The molecular weight (M_(w)) of the precursor-form wascalculated to 41 kDa, and the isoelectric point (pI) was estimated to beapproximately 4.9. Further, the amino acid composition of theaminopeptidase was estimated as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                      No.  Percent                                                    ______________________________________                                        Non-polar:                                                                      Ala 37 9.79                                                                   Val 23 6.08                                                                   Leu 29 7.67                                                                   Ile 20 5.29                                                                   Pro 14 3.70                                                                   Met  3 0.79                                                                   Phe 19 5.03                                                                   Trp  2 0.53                                                                   Polar:                                                                        Gly 28 7.41                                                                   Ser 32 8.47                                                                   Thr 25 6.61                                                                   Cys  4 1.06                                                                   Tyr 13 3.44                                                                   Asn 11 2.91                                                                   Gln 16 4.23                                                                   Acidic:                                                                       Asp 29 7.67                                                                   Glu 25 6.61                                                                   Basic:                                                                        Lys 28 7.41                                                                   Arg  9 2.38                                                                   His 10 2.65                                                                 ______________________________________                                    

The deduced complete precursor-form of the amino acid sequence of the 35kDa enzyme is shown in SEQ ID No. 2.

Accordingly, the first aspect of the invention relates to an enzymeexhibiting aminopeptidase activity having an apparent molecular weight(M_(w)) of about 35 kDa determined by SDS-PAGE.

Mass spectrometry showed that the average mass of the recombinantaminopeptidase is in the range from 33 kDa to 35 kDa.

The isoelectric point (pI) of the enzyme was determined to be about 4.9.

The isoelectric point, pI, is defined as the pH value where the enzymemolecule complex (with optionally attached metal or other ions) isneutral, i.e. the sum of electrostatic charges (net electrostaticcharge, NEC) on the complex is equal to zero. In this sum of courseconsideration of the positive or negative nature of the electrostaticcharge must be taken into account.

In the following the terms "35 kDa aminopeptidase" and "the enzymeexhibiting aminopeptidase activity" are used interchangeably for thesingle-component enzyme of the present invention.

The enzyme exhibiting aminopeptidase activity of the invention may bederived from a number of microorganisms. The present inventors haveisolated the aminopeptidase of the invention from the filamentous fungusAspergillus oryzae A01568, which is a strain deposited at the AmericanType Culture Collection as Aspergillus oryzae 460 (FERM-P no. 1149, ATCCno. 20386, and further described in U.S. Pat. No. 3,914,436.

The enzyme exhibiting aminopeptidase activity of the invention comprisesat least one of the partial amino acid sequences shown in SEQ ID Nos. 6,7, 8, 9, and 10, respectively. SEQ ID No 11 is a peptide (5) whichoverlaps and extends the N-terminal sequence.

In the second aspect, the invention relates to a DNA constructcomprising a DNA sequence encoding said aminopeptidase, which DNAsequence comprises

a) the aminopeptidase encoding part of the DNA sequence shown in SEQ IDNo. 1, and/or the DNA sequence obtainable from E. coli DSM 9965, or

b) an analogue of the DNA sequence shown defined in a), which

i) is homologous with the DNA sequence shown in SEQ ID No. 1 and/or theDNA sequence obtainable from E. coli DSM 9965, or

ii) hybridizes with the same oligonucleotide probe as the DNA sequenceshown in SEQ ID No. 1 and/or the DNA sequence obtainable from E. coliDSM 9965, or

iii) encodes a polypeptide which is homologous with the polypeptideencoded by a DNA sequence comprising the DNA sequence shown in SEQ IDNo. 1 and/or the DNA sequence obtainable from E. coli DSM 9965, or

iv) encodes a polypeptide which is immunologically reactive with anantibody raised against the purified aminopeptidase encoded by the DNAsequence shown in SEQ ID No 1 derived from Aspergillus oryzae A01568 orobtainable from E. coli, DSM 9965.

In the present context, the "analogue" of the DNA sequence shown in SEQID No. 1 and/or the DNA sequence obtainable from E. coli DSM 9965, isintended to indicate any DNA sequence encoding an enzyme exhibitingaminopeptidase activity, which has at least one of the propertiesi)-iv).

The analogous DNA sequence

may be isolated from another or related (e.g. the same) organismproducing the enzyme exhibiting aminopeptidase activity on the basis ofany of the DNA sequences shown in SEQ ID Nos. 3-5, e.g. using theprocedures described herein, and thus, e.g. be an allelic or speciesvariant of the DNA sequence comprising the DNA sequences shown herein,

may be constructed on the basis of any of the DNA sequences shown in SEQID Nos. 3-5, e.g. by introduction of nucleotide substitutions which donot give rise to another amino acid sequence of the aminopeptidaseencoded by the DNA sequence, but which correspond to the codon usage ofthe host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions which may give rise to adifferent amino acid sequence. However, in the latter case amino acidchanges are preferably of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding oractivity of the polypeptide, small deletions, typically of one to about30 amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or a small extension that facilitates purification, suchas a poly-histidine tract, an antigenic epitope or a binding domain. Seein general Ford et al., (1991), Protein Expression and Purification 2,95-107. Examples of conservative substitutions are within the group ofbasic amino acids (such as arginine, lysine, histidine), acidic aminoacids (such as glutamic acid and aspartic acid), polar amino acids (suchas glutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine, valine), aromatic amino acids (such as phenylalanine,tryptophan, tyrosine) and small amino acids (such as glycine, alanine,serine, threonine, methionine).

It will be apparent to persons skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active enzyme. Amino acidsessential to the activity of the polypeptide encoded by the DNAconstruct of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, (1989), Science 244, 1081-1085). In the lattertechnique mutations are introduced at every residue in the molecule, andthe resulting mutant molecules are tested for biological (i.e.aminopeptidolytic) activity to identify amino acid residues that arecritical to the activity of the molecule. Sites of substrate-enzymeinteraction can also be determined by analysis of crystal structure asdetermined by such techniques as nuclear magnetic resonance,crystallography or photoaffinity labelling. See, for example, de Vos etal. (1992), Science 255, 306-312; Smith et al., (1992), J. Mol. Biol.,224, 899-904; Wlodaver et al., (1992), FEBS Lett., 309, 59-64.

It will be understood that the DNA sequences shown in SEQ ID Nos. 3-5are sequences which may be used for isolating the entire DNA sequenceencoding the aminopeptidase, e.g. the DNA sequence shown in SEQ ID No. 1and/or the DNA sequence transformed into the deposited strain E. coliDSM 9965. The term "analogue" is intended to include said entire DNAsequence, which comprises one or more of the partial sequences shown inSEQ ID Nos. 3-5 or parts thereof. The amino acid sequence (as deducedfrom the DNA sequence shown in SEQ ID No. 1) is shown in SEQ ID No. 2.

The homology referred to in i) above is determined as the degree ofidentity between the two sequences indicating a derivation of the firstsequence from the second. The homology may suitably be determined bymeans of computer programs known in the art such as GAP provided in theGCG program package (Needleman, S. B. and Wunsch, C. D., (1970), Journalof Molecular Biology, 48, p. 443-453). Using GAP with the followingsettings for DNA sequence comparison: GAP creation penalty of 5.0 andGAP extension penalty of 0.3, the coding region of the DNA sequenceexhibits a degree of identity preferably of at least at least 70%,preferably at least 80%, especially at least 90%, with the coding regionof the DNA sequence shown in SEQ ID No. 1 or the DNA sequence obtainablefrom the plasmid in E. coli DSM 9965.

The hybridization referred to in ii) above is intended to indicate thatthe analogous DNA sequence hybridizes to the same probe as the DNAsequence encoding the aminopeptidase under certain specified conditionswhich are described in detail in the Materials and Methods sectionhereinafter.

Normally, the analogous DNA sequence is highly homologous to the DNAsequence such as at least 60% homologous to the DNA sequence shown inSEQ ID No. 1 or the DNA sequence obtainable from the plasmid in E. coliDSM 9965 encoding an aminopeptidase of the invention, such as at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90% or even at least 95% homologous to said DNA sequence.

The homology referred to in iii) above is determined as the degree ofidentity between the two sequences indicating a derivation of the firstsequence from the second. The homology may suitably be determined bymeans of computer programs known in the art such as GAP provided in theGCG program package (Needleman, S. B. and Wunsch, C. D., (1970), Journalof Molecular Biology, 48, p. 443-453). Using GAP with the followingsettings for DNA sequence comparison: GAP creation penalty of 5.0 andGAP extension penalty of 0.3, the coding region of the DNA sequenceexhibits a degree of identity preferably of at least at least 70%,preferably at least 80%, especially at least 90%, with the coding regionof the DNA sequence shown in SEQ ID No. 1 or the DNA sequence obtainablefrom the plasmid in E. coli DSM 9965.

The term "derived from" in connection with property iv) above isintended not only to indicate an aminopeptidase produced by strainA01568, but also an aminopeptidase encoded by a DNA sequence isolatedfrom strain A01568 and produced in a host organism transformed with saidDNA sequence. The immunological reactivity may be determined by themethod described in the "Materials and Methods" section below.

In further aspects the invention relates to an expression vectorharbouring a DNA construct of the invention, a cell comprising the DNAconstruct or expression vector and a method of producing an enzymeexhibiting aminopeptidase activity which method comprises culturing saidcell under conditions permitting the production of the enzyme, andrecovering the enzyme from the culture.

It is also an object of the invention to provide an enzyme preparationenriched with the 35 kDa aminopeptidase of the invention.

Further, the invention provides a bread-improving or a dough-improvingcomposition comprising an enzyme exhibiting aminopeptidase activity ofthe invention. Said composition may be combined with other enzymes, suchas amylolytic enzymes, and conventional bread improving agents.

In a still further aspect the invention relates to a method forpreparing a baked product and frozen dough comprising the 35 kDaaminopeptidase of the invention.

Finally the invention relates to the use of the 35 kDa aminopeptidase ofthe invention. The enzyme of the invention or a composition of theinvention comprising such an enzyme may be used for improving theflavour, crust colour and crumb structure of baked products and toimprove the stickiness of frozen dough. The aminopeptidase of theinvention may furthermore be used advantageously in connection withproducing proteins and protein hydrolysates without bitter taste and maybe used for a number of purposes including, degradation or modificationof protein containing substances; cleaning of contact lenses,preparation of food and animal feed etc.

DETAILED DESCRIPTION OF THE INVENTION

The DNA sequence of the invention encoding an enzyme exhibitingaminopeptidase activity may be isolated by a general method involving

cloning, in suitable vectors, a DNA library from Aspergillus oryzae,

transforming suitable yeast host cells with said vectors,

culturing the host cells under suitable conditions to express any enzymeof interest encoded by a clone in the DNA library,

screening for positive clones by determining any aminopeptidase activityof the enzyme produced by such clones, and

isolating the DNA coding an enzyme from such clones.

The general method is further disclosed in WO 93/11249 the contents ofwhich are hereby incorporated by reference. A more detailed descriptionof the screening method is given in Example 3 below.

Microbial Sources

The DNA sequence coding for the aminopeptidase of the invention may forinstance be isolated by screening a cDNA library of the donor organism,and selecting for clones expressing the appropriate enzyme activity(i.e. aminopeptidase activity as defined by the ability of the enzyme tohydrolyse Leucine-7 amido-4-methylcoumarin). The appropriate DNAsequence may then be isolated from the clone by standard procedures,e.g. as described in Example 1.

The donor organism may be a fungus of the Aspergillus oryzae (ATCC no.20386) described in U.S. Pat. No. 3,914,436

The complete full length DNA sequence encoding the aminopeptidase of theinvention has been transformed into a strain of the bacteria E. coli,comprised in the expression plasmid pYES 2.0 (Invitrogen). Said bacteriahas been deposited by the inventors according to the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at the Deutshe Sammlung von Mikroorganismenund Zellkulturen GmbH., Mascheroder Weg 1b, D-38124 Braunschweig FederalRepublic of Germany, (DSM).

Deposit date: 11.05.95

Depositor's ref.: NN49001

DSM designation: E. coli DSM No. 9965

Being an International Depository Authority under the Budapest Treaty,Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH., affordspermanence of the deposit in accordance with the rules and regulationsof said treaty, vide in particular Rule 9. Access to the two depositswill be available during the pendency of this patent application to onedetermined by the Commisioner of the United States Patent and TrademarkOffice to be entitled thereto under 37 C.F.R. Par. 1.14 and 35 U.S.C.Par. 122. Also, the above mentioned deposits fulfil the requirements ofEuropean patent applications relating to microorganisms according toRule 28 EPC.

The above mentioned deposit represents a substantially pure culture ofthe isolated bacteria. The deposit is available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of the deposited strain does not constitute alicense to practice the subject invention in derogation of patent rightsgranted by governmental action.

The DNA sequence encoding the enzyme exhibiting aminopeptidase activitycan for instance be isolated from the above mentioned deposited strainby standard methods.

It is expected that a DNA sequence coding for a homologous enzyme, i.e.an analogous DNA sequence, is obtainable from other microorganisms. Forinstance, the DNA sequence may be derived by similarly screening a cDNAlibrary of another microorganism, in particular a fungus, such as astrain of an Aspergillus sp., in particular a strain of A. aculeatus orA. niger, a strain of another Trichoderma sp., in particular a strain ofT. reesei, T. viride, T. longibrachiatum or T. koningii or a strain of aFusarium sp., in particular a strain of F. oxysporum, or a strain of aHumicola sp.

Alternatively, the DNA sequence coding for an enzyme exhibitingaminopeptidase activity of the invention may, in accordance withwell-known procedures, conveniently be isolated from DNA from a suitablesource, such as any of the above mentioned organisms, by use ofsynthetic oligonucleotide probes prepared on the basis of a DNA sequencedisclosed herein. For instance, a suitable oligonucleotide probe may beprepared on the basis of any of the nucleotide sequences shown in SEQ IDNos. 3-5 or the amino acid sequence shown in SEQ ID No. 2 or anysuitable subsequence thereof.

The DNA sequence may subsequently be inserted into a recombinantexpression vector. This may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the aminopeptidase should beoperably connected to a suitable promoter and terminator sequence. Thepromoter may be any DNA sequence which shows transcriptional activity inthe host cell of choice and may be derived from genes encoding proteinseither homologous or heterologous to the host cell. The procedures usedto ligate the DNA sequences coding for the aminopeptidase, the promoterand the terminator, respectively, and to insert them into suitablevectors are well known to persons skilled in the art (cf., for instance,Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual, ColdSpring Harbor, N.Y.).

The host cell which is transformed with the DNA sequence encoding theenzyme of the invention is preferably an eukaryotic cell, in particulara fungal cell such as a yeast or filamentous fungal cell. In particular,the cell may belong to a species of Aspergillus, most preferablyAspergillus oryzae or Aspergillus niger. Fungal cells may be transformedby a process involving protoplast formation and transformation of theprotoplasts followed by regeneration of the cell wall in a manner knownper se. The use of Aspergillus as a host microorganism is described inEP 238 023 (Novo Nordisk A/S), the contents of which are herebyincorporated by reference. The host cell may also be a yeast cell, e.g.a strain of Saccharomyces, in particular Saccharomyces cerevisiae,Saccharomyces kluyveri or Saccharomyces uvarum, a strain ofSchizosaccharomyces sp., such as Schizosaccharomyces pombe, a strain ofHansenula sp. Pichia sp., Yarrowia sp. such as Yarrowia lipolytica, orKluyveromyces sp. such as Kluyveromyces lactis.

A Method of Producing an Enzyme of the Invention

In a still further aspect, the present invention relates to a method ofproducing an enzyme according to the invention, wherein a suitable hostcell transformed with a DNA sequence encoding the enzyme is culturedunder conditions permitting the production of the enzyme, and theresulting enzyme is recovered from the culture.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed aminopeptidase may conveniently be secreted into the culturemedium and may be recovered there from by well-known proceduresincluding separating the cells from the medium by centrifugation orfiltration, precipitating proteinaceous components of the medium bymeans of a salt such as ammonium sulphate, followed by chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Enzyme Preparation

In a still further aspect, the present invention relates to an enzymepreparation useful for reducing the bitterness of proteins and/orprotein hydrolysates for foodstuff.

The enzyme preparation, having been enriched with an enzyme of theinvention, may e.g. be an enzyme preparation comprising multipleenzymatic activities, such as an enzyme preparation comprising multipleenzymes for producing protein hydrolysates. The preparation to beenriched can be Flavourzyme® (available from Novo Nordisk A/S).Flavourzyme® is a protease/peptidase complex derived from Aspergillusoryzae developed for hydrolysis of proteins.

Dependent on the use for which the enzyme preparation is to be used theaminopeptidase of the invention may be combined with other enzyme asmentioned below.

In the present context, the term "enriched" is intended to indicate thatthe aminopeptidase activity of the enzyme preparation has beenincreased, e.g. with an enrichment factor of at least 1. 1, preferablebetween 1.1 and 10, more preferred between 2 and 8, especially between 4and 6, conveniently due to addition of an enzyme of the inventionprepared by the method described above.

Alternatively, the enzyme preparation enriched with an enzyme exhibitingaminopeptidase activity may be one which comprises an enzyme of theinvention as the major enzymatic component, e.g. a single-componentenzyme preparation.

The enzyme preparation may be prepared in accordance with methods knownin the art and may be in the form of a liquid or a dry preparation. Forinstance, the enzyme preparation may be in the form of a granulate or amicrogranulate. The enzyme to be included in the preparation may bestabilized in accordance with methods known in the art.

In another aspect the invention relates to a bread-improving or adough-improving composition comprising an aminopeptidase of theinvention. Said composition may further comprise enzymes selected fromthe group including amylolytic enzyme, such as α-amylase, β-amylase,maltogenic α-amylase, amyloglucosidase, acid stable amylase, and1,6-pullulanase.

Such enzymes are available from Novo Nordisk A/S as AMG™(amyloglucosidase) obtained from a strain of Aspergillus niger,Fungamyl™ (fungal amylase) obtained from a strain of Aspergillus oryzae,Novamyl™ (maltogenic amylase) obtained from a strain of Bacillusstearothermophilus.

The composition of the invention may also comprise one or moreadditional enzymes. Examples of such enzymes include a cellulase, ahemicellulase, a pentosanase (useful for the partial hydrolysis ofpentosans which increases the extensibility of the dough), a lipase(useful for modification of lipids present in the dough or doughconstituents so as to soften the dough), a peroxidase (useful forimproving the dough consistency), an oxidase, e.g. a glucose oxidase, alaccase, a xylanase, a protease (useful for gluten weakening, inparticular when using hard wheat flour).

The other enzyme components are preferably of microbial origin and maybe obtained by conventional techniques used in the art as mentionedabove.

The enzyme(s) to be used in the present invention may be in any formsuited for the use in question, e.g. in the form of a dry powder orgranulate, in particular a non-dusting granulate, a liquid, inparticular a stabilized liquid, or a protected enzyme. Granulates may beproduced, e.g. as disclosed in U.S. Pat. No. 4,106,991 and U.S. Pat. No.4,661,452 (both to Novo Industri A/S), and may optionally be coated bymethods known in the art. Liquid enzyme preparations may, for instance,be stabilized by adding nutritionally acceptable stabilizers such as asugar, a sugar alcohol or another polyol, lactic acid or another organicacid according to established methods. Protected enzymes may be preparedaccording to the method disclosed in EP 238,216.

Normally, for inclusion in pre-mixes or flour it is advantageous thatthe enzyme(s) is/are in the form of a dry product, e.g. a non-dustinggranulate, whereas for inclusion together with a liquid it isadvantageously in a liquid form.

In addition or in an alternative to other enzyme components, thedough-improving and/or bread-improving composition may comprise aconventionally used baking agent, e.g. one or more of the followingconstituents:

A milk powder (providing crust colour), gluten (to improve the gasretention power of weak flours), an emulsifier (to improve doughextensibility and to some extent the consistency of the resultingbread), granulated fat (for dough softening and consistency of bread),an oxidant (added to strengthen the gluten structure; e.g. ascorbicacid, potassium bromate, potassium iodate or ammonium persulfate), anamino acid (e.g. cysteine), a sugar, and salt (e.g. sodium chloride,calcium acetate, sodium sulfate or calcium sulphate serving to make thedough firmer), flour or starch. Such components may also be addeddirectly to the dough in accordance with a method of the invention.

Examples of suitable emulsifiers are mono- or diglycerides, diacetyltartaric acid esters of mono- or diglycerides, sugar esters of fattyacids, polyglycerol esters of fatty acids, lactic acid esters ofmonoglycerides, acetic acid esters of monoglycerides, polyoxyethylenestearates, phospholipids and lecithin.

The bread-improving and/or dough improving composition of the inventionis typically included in the dough in an amount corresponding to0.01-5%, in particular 0.1-3%.

In accordance with the method of the invention, in which an enzyme withaminopeptidase activity of the invention, optionally in combination withother enzymes as described above, is used for the preparation of doughand/or baked products, the enzyme(s) may be added as such to the mixturefrom which the dough is made or to any ingredient, e.g. flour, fromwhich the dough is to be made. Alternatively, the enzyme(s) may be addedas a constituent of a dough-improving and/or a bread-improvingcomposition as described above, either to flour or other doughingredients or directly to the mixture from which the dough is to bemade.

The dosage of the enzyme(s) to be used in the method of the presentinvention should be adapted to the nature and composition of the doughin question as well as to the nature of the enzyme(s) to be used.Normally, the enzyme preparation is added in an amount corresponding to0.01-1000 mg enzyme protein per kg of flour, preferably 0.1-100 mgenzyme protein per kg of flour, more preferably 0.1-10 mg enzyme proteinper kg of flour.

In terms of enzyme activity, the appropriate dosage of a given singlecomponent enzyme with aminopeptidase activity, optionally in combinationwith other enzyme(s), for exerting a desirable improvement of flour orcrust colour of a baked product will depend on the enzyme(s) and theenzyme substrate(s) in question. The optimal dosage may vary dependenton the flour or yeast types and baking process. The skilled person maydetermine a suitable enzyme unity dosage on the basis of methods knownin the art.

However, according to the present invention the enzyme exhibitingaminopeptidase activity of the invention is added in an amountcorresponding 30 to 1000 LAPU, preferably 50 to 500 LAPU, especially 80to 300 LAPU, such as about 100 LAPU per kg of flour. LAPU is definedbelow.

Amylolytic enzymes are normally added in from 1 to 50 FAU per kg flour.One FAU (Fungal α-Amylase Unit) is the amount of enzyme which breaksdown 5.26 g starch (Merck, Amylum solubile Erg. B.6, BAch 9947275) perhour using Novo Nordisk's standard method for determination of αamylaseactivity. A detailed description of Novo Nordisk's method (AF 216) ofanalysis is available on request.

Maltogenic amylase are normally added in from 1 to 1000 MANU per kgflour (Maltogenic Amylase Novo Units). One MANU is defined as the amountof enzyme which, under standard conditions, hydrolyzes i micromole ofmaltotriose per minute. The analytic method (AF 203) is available onrequest.

When one or more additional enzyme activities are to be added inaccordance with the method of the invention, these activities may beadded separately or together with the singes component enzyme withexopeptidase activity, optionally as constituent(s) of thebread-improving and/or dough-improving composition of the invention. Theother enzyme activities may be any of the above described enzymes andmay be dosed in accordance with established baking practice.

As mentioned above the enzyme exhibiting aminopeptidase activity,optionally in combination with other enzyme(s) as described above, isadded to any mixture of dough ingredients, to the dough, or to any ofthe ingredients to be included in the dough, in other words theenzyme(s) may be added in any step of the dough preparation and may beadded in one, two or more steps, where appropriate.

The handling of the dough and/or baking is performed in any suitablemanner for the dough and/or baked product in question, typicallyincluding the steps of kneading the dough, subjecting the dough to oneor more proofing treatments, and baking the product under suitableconditions, i.e. at a suitable temperature and for a sufficient periodof time. For instance, the dough may be prepared by using a normalstraight dough process, a sour dough process, an overnight dough method,a low-temperature and long-time fermentation method, a frozen doughmethod, the Chorleywood Bread process, or the Sponge and Dough process.

The dough and/or baked product prepared by the method of the inventionare normally based on wheat meal or flour, optionally in combinationwith other types of meal or flour such as corn flour, rye meal, ryeflour, oat flour or meal, soy flour, sorghum meal or flour, or potatomeal or flour.

In the present context the term "baked product" is intended to includeany product prepared from dough, either of a soft or a crisp character.Examples of baked products, whether of a white, light or dark type,which may advantageously be produced by the present invention are bread(in particular white, whole-meal or rye bread), typically in the form ofloaves or rolls, French baguette-type bread, pita bread, tacos, cakes,pan-cakes, biscuits, crisp bread and the like.

The dough of the invention may be of any of the types discussed above,and may be fresh or frozen.

The preparation of frozen dough is described by K. Kulp and K. Lorenz in"Frozen and Refrigerated Doughs and Batters". When using theaminopeptidase of the invention for frozen bread the flavour, the crustcolour and the crispiness are improved.

From the above disclosure it will be apparent that the dough of theinvention is normally a leavened dough or a dough to be subjected toleavening. The dough may be leavened in various ways such as by addingsodium bicarbonate or the like or by adding a leaven (fermenting dough),but it is preferred to leaven the dough by adding a suitable yeastculture such as a culture of Saccharomyces cerevisiae (baker's yeast).Any of the commercially available S. cerevisiae strains may be employed.

As mentioned above, the present invention further relates to a pre-mix,e.g., in the form of a flour composition, for dough and or bakedproducts made from dough, which pre-mix comprises an enzyme exhibitingaminopeptidase activity of the invention and optionally other enzymes asspecified above. The pre-mix may be prepared by mixing the relevantenzyme(s) or a bread-improving and/or dough-improving composition of theinvention comprising the enzyme(s) with a suitable carrier such asflour, starch, a sugar or a salt. The pre-mix may contain otherdough-improving and/or bread-improving additives, e.g., any of theadditives, including enzymes, mentioned above.

Use of the 35 kDa Aminopeptidase of the Invention

Use of the Enzyme of the Invention for Preparing Baked Products

The enzyme or an enzyme preparation of the invention may be used inbaking, e.g. in order to weaken the gluten components of flour so as toobtain a softening of so-called hard flour.

However, it has surprisingly been found that the aminopeptidase of theinvention does not degrade the network of the gluten which is normallyobserved when proteases are used for preparing baked products.Consequently, the dough characteristics and crumb structure are notaffected.

Further, when adding the 35 kDa aminopeptidase of the invention to thedough or dough ingredients, when preparing baked products, the flavourcrust colour and/or the crumb structure and/or the crust colour of abaked product will be substantially improved, as shown in Example 13.

Further, the enzyme of the invention improves the dough stickiness.

The addition of the enzyme of the invention results in baked productshaving a flavour of yeasty type, providing a "freshly baked" breadsmell. This make the invention of particular interest for thesponge-dough-system, in which an addition of the enzyme can reduce thesponge fermentation time without a concomitant loss of yeasty flavour,and in the no-time-dough process (most European processes), in which theenzyme can provide a yeasty flavour which is otherwise normally lackingproducts prepared from such processes.

Without being limited to any theory it is presently believed thatfurther improved flavour and/or crust colour of a baked product may beobtained when a single component enzyme with exopeptidase activity isused in combination with an amylolytic enzyme, in particular anamylolytic enzyme which is capable of liberating reducing sugarmolecules from flour or other constituents of the dough. The increasedamount of reducing sugars in the dough provides an increase in Maillardreactions taking place during baking thereby further improving theflavour and crust colour of the baked product.

Use of the Enzyme of the Invention for Reducing Bitter Taste

The enzyme or enzyme preparation of the invention may be used forreducing the bitterness of proteins and/or protein hydrolysate forfoodstuff.

Also contemplated according to the invention is the production of freeamino acids from proteins and/or protein hydrolysates. In the case ofthe free amino acid are glutamine acid it enhances the flavour of foodproducts.

Said protein or protein hydrolysate may be of animal or vegetableorigin.

In an embodiment of the invention the protein to be hydrolysed is caseinor soy protein.

The protein may be use for producing foodstuff such as cheese andfoodstuff containing cocoa.

Even though the aminopeptidase and enzyme preparations enriched with anenzyme of the invention may be used especially advantageously inconnection with producing proteins or protein hydrolysates withoutbitter taste, the aminopeptidase of the invention can be used for anumber of industrial applications, including degradation or modificationof protein containing substances, such cell walls. Some proteins, likeextensins, are components of plant cell walls. Aminopeptidases willtherefore facilitate the degradation or modification of plant cellwalls.

The dosage of the enzyme preparation of the invention and otherconditions under which the preparation is used may be determined on thebasis of methods known in the art.

Extraction of Oil from Plants

The enzyme preparation according to the invention may be useful forextraction of oil from plant sources like olives and rape or forproduction of juice from different fruits like apples, pears and citrus.It may also be useful in the wine industry, especially in the white wineindustry, to prevent haze formation. Furthermore, it may be used tomodify and degrade proteins, e.g. in order to reduce the viscositycaused or partially caused by proteins, or to facilitate fermentativeprocesses where proteins are involved, or it may be used to improve thedigestibility of proteins and other nutrients.

The Use for Preparing Food and Feed

The aminopeptidase preparation may also be used in the food and feedindustry to improve the digestibility of proteins. For instance, theenzyme or enzyme preparation may be added to animal feed or may be usedto process animal feed, in particular feed for piglets or poultry.

Further the enzyme or enzyme preparation of the invention may be usefulto make protein hydrolysates from, e.g., vegetable proteins like soy,pea, lupin or rape seed protein, milk like casein, meat proteins, orfish proteins. The aminopeptidase may be used for protein hydrolysatesto improve the solubility, consistency or fermentability, to reduceantigenicity or for other purposes to make food, feed or medicalproducts. The aminopeptidase may be used alone or together with otheraminopeptidases or together with other enzymes like exopeptidases. Theuse of the aminopeptidase of the invention together with exopeptidaserich enzyme preparations will improve the taste of the proteinhydrolysates.

Furthermore, the enzyme or enzyme preparation may be used in theprocessing of fish or meat, e.g. to change texture and/or viscosity.

Use in Brewing Processes

The enzyme preparation may also be used to facilitate fermentativeprocesses, like yeast fermentation of barley, malt and other rawmaterials for the production of e.g. beer.

Use for Making Protoplast

The enzyme preparation may be useful for making protoplasts from fungi.

Use for the Production of Peptides

The enzyme preparation may be useful for production of peptides fromproteins, where it is advantageous to use a cloned enzyme essentiallyfree from other proteolytic activities.

Use for Degradation of Proteins

Further, the aminopeptidase preparation can be used to degrade proteinin order to facilitate purification of or to upgrade different products,like in purification or upgrading of gums, like guar gum, xanthan gum,degumming of silk, or improvement of the quality of wool.

Use for Cleaning Contact Lenses

Further the enzyme or enzyme preparation may be used for cleaning ofcontact lenses.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

Methods and Materials

Materials

Donor Organism

Aspergillus oryzae A01568 (described in U.S. Pat. No. 3,914,436)

Host organism

Escherichia coli MC1061 (Meissner et al., (1987), Proc. Natl. Acad. Sci.U.S.A., 84, 4171-4176: cDNA library strain

Saccharomyces cerevisiae W3124 (van den Hazel et al., (1992), Eur. J.Biochem., 207, 277-283): Activity screening strain.

Schizosaccharomyces pombe: Broker et al., (1989), FEBS Letters, 248,105-110.

Other Organisms

Aspergillus oryzae A1560 (Christensen et al. (1988), Bio/technology 6,1419-1422).

Plasmids

pYES 2.0: Transformation vector (Invitrogen).

pHD414: Aspergillus expression vector is a derivative of the plasmidp775 (described in EP 238.023). The construction of the pHD414 isfurther described in WO 93/11249. pHD414 contains the A. nigerglucoamylase terminator and the A. oryzae TAKA amylase promoter.

pHD423 is a derivative of pHD414 (described in WO 94/20611) with a newpolylinker.

pUC18: Expression vector (Sambrook et al., (1989), Molecular Cloning: ALaboratory Manual, 2. ed.; Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.)

pC1EXP3: plasmid comprising the 1.4 kb cDNA insert encoding the 35 kDaaminopeptidase of the invention. (See Example 3 and SEQ ID NO 1)

pC1EXP4: plasmid comprising a cDNA sequence, which is 120 bp shorterthat the pC1EXP3 1.4 kb cDNA insert. (see Example 10 and SEQ ID NO 12)

p3SR2: A. nidulans amdS+ gene carrying plasmid (Christensen et al.,(1988), Bio/Technology 6, 1419-1422)

pP1: yeast expression vector, which is an E. coli/S. pombe shuttlevector containing the ADH promoter and URA 3 as selective marker (Brokeret al., (1989), FEBS Letters. 248, 105-110).

Primers

Universal pUC primers (Sambrook et al., (1989), supra)

Deduced Primer Sequences Used in PCR Reactions

s2:

5'- GAR ACI GTI CAR AAY CTI AT -3' (SEQ ID NO:13)

s3:

5'- GAY AAR AAR AAY TTY GAW ACI GT -3' (SEQ ID NO:14)

as1:

5'- TCI ACR TTR TCI GTI ATI ATY TCI AT -3' (SEQ ID NO:15)

(s=sense, as=anti-sense)

A=Adenine

G=Guanine

C=Cytosine

T=Thymine

I=Deoxyinosine

Y=C or T

R=A or G

W=A or T

Forward and Reverse pYES Primers (Invitrogen)

Enzymes

Lysine-specific protease

Novozym® 234 (Novo Nordisk A/S)

Alcalase® (Novo Nordisk A/S)

Neutrase® (Novo Nordisk A/S)

Peptides of the 35 kDa Aminopeptidase

(see SEQ ID No. 6-11)

N-terminal

Direct N-terminal sequencing of authentic and recombinant aminopeptidaserevealed the same N-terminal amino acid sequence for the two enzymesshowing that the recombinant enzyme is proteolytically processedidentical to the authentic enzyme. The N-terminal sequence found was

Tyr-Pro-Asp-Ser-Val-Gln-His-Xaa-Glu-Thr-Val-Gln-Asn-Leu-Ile-s2→

Lys-Ser-Leu-Asp-Lys-Lys-Asn-Phe-Glu-Thr-Val-Leu-Gln-Pro- (SEQ IDNO:10)s3→

(Xaa is a glycosylated Asn-residue)

The following peptide sequence was obtained from peptides derived from aS-carboxymethylated sample of the aminopeptidase by cleavage with alysyl-specific protease.

Peptide 1

Tyr-Pro-Asp-Ser-Val-Gln-His-Xaa-Glu-Thr-Val-Gln-Asn-Leu-Ile-Lys (SEQ IDNO:6)

Peptide 2

Gly-Val-Thr-Val-Glu-Pro-Phe-Lys (SEQ ID NO:7)

Peptide 3

Val-Ile-Val-Asp-Ala-Tyr-Cys-Thr-Ile-Pro-Thr-Val-Asp-Ser-Lys (SEQ IDNO:8)

Peptide 4

Gly-Thr-Thr-Asp-Ala-Gly-Lys-Pro-Glu-Ser-Ile- Glu-Ile-Ile-Thr-←as1

Asp-Asn-Val-Asp-Glu-Asn-Leu-Thr-Lys (SEQ ID NO:9)

Peptide 5

Asn-Phe-Glu-Thr-Val-Leu-Gln-Pro-Phe-Ser-Glu-Phe-His-Asn-Arg-Tyr-Tyr-Lys(SEQ ID NO:11)

(Overlaps and Extends the N-terminal Sequence)

Media and Other Materials

STC: 1.2 M sorbitol, 10 mM Tris-HCl, pH=7.5., 10 mM CaCl₂

BSA (Sigma, type H25)

Leucine-7 amido-4-methylcoumarin (Sigma)

PEG 4000 (polyethylene glycol, molecular weight=4,000) (BDH, England)

Sequenase®Kit (United Stated Biochemical, USA)

Hybond-N nylon membrane (Amersham, USA)

Q-sepharose (Pharmacia Tm)

Superdex 200 Tm

Amicon membrane

VG Analytical TofSpec

α-Cyano-4-hydroxycinnamic acid (Aldrich, Steinheim, Germany)

YPD: 10 g yeast extract, 20 g peptone, H₂ O to 810 ml. Autoclaved, 90 ml20% glucose (sterile filtered) added.

10×Basal salt: 66.8 g yeast nitrogen base, 100 g succinic acid, 60 gNaOH, H₂ O ad 1000 ml, sterile filtered.

SC-URA: 90 ml 10×Basal salt, 22.5 ml 20% casamino acids, 9 ml 1%tryptophan, H₂ O ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml20% glucose or 20% galactose added. SC-H broth: 7.5 g/l yeast nitrogenbase without amino acids, 11.3 g/l succinic acid, 6.8 g/l NaOH, 5.6 g/lcasamino acids without vitamins, 0.1 g/l tryptophan. Autoclaved for 20min. at 121° C. After autoclaving, 10 ml of a 30% galactose solution, 5ml of a 30% glucose solution and 0.4 ml of a 5% threonine solution wereadded per 100 ml medium.

SC-H agar: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/lsuccinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins,0.1 g/l tryptophan, and 20 g/l agar (Bacto). Autoclaved for 20 min. at121° C. After autoclaving, 55 ml of a 22% galactose solution and 1.8 mlof a 5% threonine solution were added per 450 ml agar.

YNB-1 agar: 3.3 g/l KH₂ PO₄, 16.7 g/l agar, pH adjusted to 7. Autoclavedfor 20 min. at 121° C. After autoclaving, 25 ml of a 13.6% yeastnitrogen base without amino acids, 25 ml of a 40% glucose solution, 1.5ml of a 1% L-leucine solution and 1.5 ml of a 1% histidine solution wereadded per 450 ml agar.

YNB-1 broth: Composition as YNB-1 agar, but without the agar.

Minimal plates: (Cove Biochem.Biophys.Acta 113 (1966) 51-56) containing1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl

Whey protein hydrolysate: Whey hydrolysed with Alcalase® and Neutrase®was diluted with water until the protein content was 8% (w/w) of thetotal solution.

Methods

RNA isolation

The total RNA was prepared, from frozen, powdered mycelium of A. oryzaeA01568, by extraction with guanidinium thiocyanate followed byultracentrifugation through a 5.7 M CsCl cushion (Chirgwin et al.,(1979), Biochemistry 18, 5294-5299). The poly(A)⁺ RNA was performed byoligo(dT)-cellulose affinity chromatography (Aviv, H, and Leder, P.,(1972), Proc. Natl. Acad. Sci. U.S.A. 69, 1408-1412).

cDNA Synthesis

Double-stranded cDNA was synthesized from 5 μg of Aspergillus oryzaepoly(A)⁺ RNA as described by Kofod et al., J. of Biol. Chem., (1994),269, 29182-29189, except that 25 ng of random hexanucleotide primers(Pharmacia, Sweden) were included in the first strand synthesis.

Construction of cDNA Library

The cDNA library was constructed as described by Kofod et al., J. ofBiol. Chem., (1994), 269, 29182-29189.

Transformation of Saccharomyces Cerevisiae

To ensure that all the bacterial clones were tested in yeast, a numberof yeast transformants 5 times larger than the number of bacterialclones in the original pools was set as the limit.

One μl aliquots of purified plasmid DNA (100 ng/μl) from individualpools were electroporated (200Ω, 1.5 kV, 24 μF) into 40 μl of competentS. cerevisiae cells (OD600=1.5 in 500 ml YPD, washed twice in colddistilled water, once in cold 1 M sorbitol, resuspended in 0.5 ml 1 Msorbitol, Becker & Guarante, 1991). After addition of 1 ml 1 M coldsorbitol, 80 μl aliquots were plated on SC+glucose-uracil to give250-400 c.f.u./plate and incubated at 30° C. for 3-5 days.

Transformation of Schizosaccharomyces pombe

Schizosaccharomyces pombe was transformed as described by Broker, (1987)BioTechniques, 5, 51-517.

Purification of 35 kDa Aminopeptidase from Aspergillus oryzae

One gram freeze dried powder of the fermentation supernatant ofAspergillus oryzae A01568 was dissolved in 100 ml Tris-acetate buffer(25 mM pH 8). Ionic strength was 2 mSi. The suspension was filteredthrough 45μ millipore filter. The filtered solution was applied on a 200ml anion exchange chromatography column packed with Q-sepharose, whichwas equilibrated with the Tris-acetate buffer. Alkaline protease whichis a major endoprotease with isoelectric point of 8 was collected ineffluent. The column was washed with the Tris-acetate buffer until nomore UV absorbing material was present in effluent.

The bound proteins were eluted with linear salt gradient using 0 to 0.5M NaCl in the Tris-acetate buffer (pH 8) using 10 column volume with aflow rate of 4 ml/minutes. Fractions containing aminopeptidase activity(see below) were pooled and dialyzed against Tris-acetate buffer (25 mM,pH 6).

The dialyzed pool containing activity was adjusted to pH 6 and ionicstrength to 2 mSi and applied on 50 ml High performance Q-sepharosecolumn, equilibrated with 25 mM Tris-acetate buffer pH 6, for anionexchange chromatography. The column was then washed until the UVabsorbing material in effluent was under 0.05 at 280 nm. Bound activitywas then eluted with 20 column volume linear salt gradient, from 0 to0.5 M NaCl at a flow rate of 2 ml/minutes. Fractions containingaminopeptidase activity were pooled and concentrated by ultrafiltration,using 50 mM sodium-acetate buffer (pH 6).

Two ml of the concentrated pool containing aminopeptidase activity wasapplied on Superdex 200 Tm column equilibrated with 50 mM sodium acetatebuffer (pH 6) containing 0.1 M NaCl. The gel filtration was carried outusing a flow rate of 0.5 ml/minutes. Samples containing aminopeptidaseactivity were pooled and concentrated by ultrafiltration using Amiconmembrane with a cut-off-value of 10 kDa.

Amino Acid Sequence Determination of N-terminal and Internal Peptides ofthe Aspergillus oryzae Aminopeptidase

S-carboxymethylated samples of the purified native aminopeptidase isdigested with lysyl-specific protease, and the resulting peptides areseparated by reverse phase high pressure liquid chromatography (HPLC)and sequenced as described by Matsudaira. A Practical Guide to Proteinand peptide Purification for Microsequencing, 3-88, Academic Press Inc,San Diego, Calif., in an Applied Biosystems 473A sequencer according tothe manufacturer's instructions (Applied Biosystems).

Reagents and solvents for amino acid sequencing are from AppliedBiosystems (Foster City, Calif.).

Designing of PCR Primers

The PCR primers were designed as described by Kofod et al., J. of Biol.Chem., (1994), 269, 29182-29189.

Generation of a cDNA Probe for an Aminopeptidase Using PCR

One μg of double stranded plasmid DNA from a cDNA library pool was PCRamplified using 500 pmol of each of the designed primers in combinationswith 500 pmol of pYES 2.0 polylinker primer (forward and reverse), a DNAthermal cycler (Landgraf, Germany) and 2.5 units of Taq polymerase(Perkin-Elmer).

Thirty cycles of PCR were performed using a cycle profile ofdenaturation at 94° C. for 1 minute, annealing at 55° C. for 2 minutes,and extension at 72° C. for 3 minutes.

Dideoxy Chain-termination Method

The method was carried out as described by Sanger et al., (1977), Proc.Natl. Acad. Sci. U.S.A. 74, 5463-5467, using the Sequenase®Kit anduniversal pUC primers.

Characterization of Positive cDNA Clones by Southern Blot Analysis

The positive clones were characterized by the use of Southern blothybridization using the 0.5 kb random-primed ³² P-labelled PCR-productor 35 kDa aminopeptidase as probe. The hybridizations were carried outin 2×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 100 μg/ml denaturedsalmon sperm DNA for 48 hours at 65° C. followed by washing at highstringency in 2×SSC (2×15 minutes), 2×SSC, 0.5% SDS (30 minutes),0.2×SSC, 0.5% SDS (30 minutes) and finally in 2×SSC (15 minutes), at 65°C. (Sambrook et al. (1989), supra).

Electrophoresis

Electrophoresis was performed on a 0.7% agarose gel from SeaKem, FMC.

Capillary Blotting

Capillary blotting method is described by Sambrook et al., (1989),supra) using 10×SSC as transfer buffer

High Stringency Washes of Hybridized Clones

Washing was carried out in 2×15 minutes in 2×SSC, 2×30 minutes in0.1×SSC, 0.5% SDS and 15 minutes in 2×SSC, at 65° C.

Transformation of Aspergillus oryzae

Transformation of Aspergillus oryzae was carried out as described byChristensen et al., (1988), Biotechnology 6, 1419-1422.

Construction of the Aminopeptidase Expression Cassette for Aspergillus

Plasmid DNA was isolated from the positive E. coli clones using standardprocedures and analyzed by restriction enzyme analysis. The cDNA insertwas excised using appropriate restriction enzymes and ligated into anAspergillus expression vector.

Transformation of Aspergillus oryzae or Aspergillus niger

(General Procedure)

100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory, 1981) is inoculated with spores of A. oryzae or A.niger and incubated with shaking at 37° C. for about 2 days. Themycelium is harvested by filtration through miracloth and washed with200 ml of 0.6 M MgSO₄. The mycelium is suspended in 15 ml of 1.2 MMgSO₄. 10 mM NaH₂ PO₄, pH=5.8. The suspension is cooled on ice and 1 mlof buffer containing 120 mg of Novozym® 234 is added. After 5 minutes 1ml of 12 mg/ml BSA is added and incubation with gentle agitationcontinued for 1.5-2.5 hours at 37° C. until a large number ofprotoplasts is visible in a sample inspected under the microscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayered with 5 ml of 0.6M sorbitol, 100 mMTris-HCl, pH=7.0. Centrifugation is performed for 15 minutes at 100 gand the protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC are added to the protoplast suspension and the mixture iscentrifugated for 5 minutes at 1000 g. The protoplast pellet isresuspended in 3 ml of STC and repelleted. This is repeated.

Finally the protoplasts are resuspended in 0.2-1 ml of STC.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriateDNA in 10 μl of STC. Protoplasts are mixed with p3SR2 (an A. nidulansamdS gene carrying plasmid). The mixture is left at room temperature for25 minutes. 0.2 ml of 60% PEG 4000. 10 mM CaCl₂ and 10 mM Tris-HCl, pH7.5 is added and carefully mixed (twice) and finally 0.85 ml of the samesolution is added and carefully mixed. The mixture is left at roomtemperature for 25 minutes, spun at 2500 g for 15 minutes and the pelletis resuspended in 2 ml of 1.2 M sorbitol. After one more sedimentationthe protoplasts are spread on the appropriate plates. Protoplasts arespread on minimal plates to inhibit background growth. After incubationfor 4-7 days at 37° C. spores are picked and spread for single colonies.This procedure is repeated and spores of a single colony after thesecond re-isolation is stored as a defined transformant.

Purification of the Aspergillus oryzae Transformants

Aspergillus oryzae colonies are purified through conidial spores onAmdS⁺ -plates (+0.01% Triton X-100) and growth in YPM for 3 days at 30°C.

Identification of Aminopeptidase Positive Aspergillus oryzaeTransformants

The supernatants from the Aspergillus oryzae transformants were assayedfor aminopeptidase on agar plates overlayered with 60 μg/ml of Leucine-7amido-4-methylcoumarin. Positive transformants were identified byanalyzing the plates by fluorescence under UV-light after 5 minutes to 2hours incubation at 30° C.

SDS-PAGE Analysis

SDS-PAGE analysis of supernatant from an Aspergillus oryzaeaminopeptidase producing transformant. The transformant was grown in 5ml YPM for three days. 10 μl of supernatant was applied to 12%SDS-polyacrylamide gel which was subsequently stained with CoomassieBrilliant Blue.

Mass Spectrometry

Mass spectrometry is done using matrix assisted laser desorptionionisation time-of flight mass spectrometry in a VG Analytical TofSpec.For mass spectrometry 2 μl of sample is mixed with 2 μl saturated matrixsolution (α-cyano-4-hydroxycinnamic acid in 0.1% TFA:acetonitrile(70:30)) and 2 μl of the mixture spotted onto the target plate. Beforeintroduction into the mass spectrometer the solvent was removed byevaporation. Samples are desorbed and ionised by 4 ns laser pulses (337nm) at threshold laser power and accelerated into the field-free flighttube by an accelerating voltage of 25 kV. Ions were detected by a microchannel plate set at 1850 V. The spectra is calibrated externally withproteins of known mass.

Immunological Cross-reactivity

Antibodies to be used in determining immunological cross-reactivity maybe prepared by use of a purified aminopeptidase. More specifically,antiserum against the aminopeptidase of the invention may be raised byimmunizing rabbits (or other rodents) according to the proceduredescribed by N. Axelsen et al. in: A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter23, or A. Johnstone and R. Thorpe, Immunochemistry in Practice,Blackwell Scientific Publications, 1982 (more specifically pp. 27-31).Purified immunoglobulins may be obtained from the antisera, for exampleby salt precipitation ((NH₄)₂ SO₄), followed by dialysis and ionexchange chromatography, e.g. on DEAE-Sephadex. Immunochemicalcharacterization of proteins may be done either by Outcherlonydouble-diffusion analysis (O. Ouchterlony in: Handbook of ExperimentalImmunology (D. M. Weir, Ed.), Blackwell Scientific Publications, 1967,pp. 655-706), by crossed immunoelectrophoresis (N. Axelsen et al.,supra, Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsenet al., Chapter 2).

Southern Blot Analysis

Genomic DNA from A. oryzae is isolated according to Yelton et al.,(1984), Proc. Natl. Acad. Sci. U.S.A. 81., p. 1470-1474, and digested tocompletion with BamHI, BglII, EcoRI and HindIII (10 μg/sample),fractionated on a 0.7% agarose gel, denatured and blotted to a nylonfilter (Hybond-N) using 1033 SSC as transfer buffer (Southern, E. M.,(1975), J. Mol. Biol. 98, p. 503-517). The aminopeptidase cDNA is ³²P-labeled (>1×10⁹ cpm/μg) by random-priming and used as a probe inSouthern analysis. The hybridization and washing conditions are asdescribed in RNA gel blot analysis. The filter is autoradiographed at-80° C. for 12 hours.

RNA Gel Blot Analysis

Poly(A)⁺ RNA (1 μg) from A. oryzae is electrophoresed in 1.2%agarose-2.2 M formaldehyde gels (Thomas, P. S., (1983) Methods Enzymol.100, pp. 255-2663) and blotted to a nylon membrane (Hybond-N) with10×SSC as transfer buffer. The aminopeptidase cDNA is ³² P-labeled(>1×10⁹ cpm/μg) by random priming and hybridized to the membrane for18-20 hours at 65° C. in 5×SSC, 5×Denhardt's solution, 0.5% SDS (w/v)and 100 μg/ml denatured salmon sperm DNA. The filter is washed in 5×SSCat 65° C. (2×15 minutes), 2×SSC, 0.5% SDS (1×30 minutes), 0.2×SSC, 0.5%SDS (1×30 minutes), and 5×SSC (2×15 minutes). The filter isautoradiographed at -80° C. for 12 hours.

Determination of Aminopeptidase Activity (LAPU)

One LAPU is defined as the amount of enzyme which hydrolyzes 1 μmole ofL-leucine-p-nitroanilide per minute using the method described in AF298/1-GB (available on request from Novo Nordisk A/S).

% DH Determination Based on TMBS Analysis

The extent of protein hydrolysis may be determined by the degree ofhydrolysis achieved. In the context of this invention, the degree ofhydrolysis (DH) is defined by the following formula: ##EQU1##

h is the number of peptide bonds hydrolysed and h_(total) is the totalnumber of peptide bonds in the protein. h_(total) is dependent on thetype of raw material, whereas h can be expressed as a function of meqvleucine NH₂, measured by for instance TNBS-analyses

Determination of % DH is described in EF-9415317 (available on requestfrom Novo Nordisk A/S)

Testing of Doughs and Breads

According to the present invention the effect of adding a singlecomponent enzyme with aminopeptidase activity may be tested in doughsand breads by using the following method:

Preparation of Breads

Procedure

1. Dough mixing (Spiral mixer)

3 min. at 700 RPM

5 min. at 1400 RPM

the mixing time is predetermined and adjusted by a skilled baker basedon the flour used so as to obtain an optimum dough consistence under thetesting conditions used.

2. 1st proof: 30° C.-80% RH, 15 min.

3. Scaling and shaping;

4. Resting for 5 minutes at ambiant temperature;

5. Final proof: 32° C.-80% RH, 45 minutes for rolls, 55 minutes forbread;

6. Baking: 225° C., 22 minutes for rolls and 30 minutes for loaf.

Evaluation of Dough and Baked Products

Dough and baked products may be evaluated as follows:

Loaf specific Volume

The mean value of 4 loaves volume are measured using the traditionalrape seed method. The specific volume is calculated as volume ml per gbread. The specific volume of the control (without enzyme) is defined as100. The relative specific volume index is calculated as: ##EQU2##

The dough stickiness and crumb structure may be evaluated visuallyaccording to the following scale:

    ______________________________________                                        Dough stickiness:                                                                              almost liquid                                                                            1                                                    too sticky 2                                                                  sticky 3                                                                      acceptable 3.5                                                                normal 4                                                                      dry 5                                                                        Crumb structure: very poor 1                                                   poor 2                                                                        non-uniform 3                                                                 uniform/good 4                                                                very good 5                                                                ______________________________________                                    

Shock Test

After the second proof a pan containing the dough is dropped from aheight of 20 cm. The dough is baked and the volume of the resultingbread is determined.

    ______________________________________                                        Yeasty Flavour  as control   3                                                   slightly improved 3.5                                                         Improved 4                                                                   Crumb colour The crumb colour is                                               determined visually                                                        ______________________________________                                    

EXAMPLES Example 1

Construction of cDNA Library

Total RNA was extracted from Aspergillus oryzae A01568. Poly(A)+RNA wasisolated by oligo(dT)-cellulose affinity chromatography and doublestranded cDNA (ds cDNA) was synthesized.

A cDNA library from Aspergillus oryzae A01568 consisting of 3.5×10⁶clones was constructed into the yeast expression vector pYES 2.0.

Example 2

Amplification and Characterization of cDNA Clones

The aminopeptidase was purified from Aspergillus oryzae A01568 asdescribed above in the section "Materials and Methods".

A long NH₂ -terminal sequence and four internal sequences (includingPeptide 1 to 5) of the aminopeptidase were obtained by digestion ofS-carboxymethylated purified protein with a lysyl-specific protease.

Based on these sequences three primers were synthesized (as1, s2 and s3,respectively). Double stranded cDNA (ds cDNA) was used as template inthe PCR amplification experiment as described above in the section"Materials and Methods".

Analysis of the resulting PCR-products revealed a 0.5 kb fragment withone primer pair (primer s3 and primer as1).

The PCR-fragment was sub-cloned into SmaI-cut dephosphorylated pUC18vector and sequenced from both ends using the Dideoxy chain-terminationmethod as described above in the section "Material and Methods".

In addition to the primer encoded residues, the sequence of Peptide 2obtained from the purified aminopeptidase, aligned with the deducedamino acid sequence, confirmed that the desired cDNA species had beenspecifically PCR amplified.

Example 3

Screening of the cDNA Library for Clones Encoding the Aminopeptidasefrom Aspergillus oryzae

Approximately 10,000 colonies from the cDNA library from Aspergillusoryzae A01568 were screened by colony hybridization as described above.This yielded positive clones with inserts ranging from 650 bp to 1.5 kb.

The positive clones were analyzed by Southern blot analysis as describedabove in the section "Materials and Methods".

Purified plasmid DNA (about 1 μg) from the aminopeptidase cDNA cloneswas digested to completion by HindIII and XbaI to release the cDNAinserts from the pYES vector. The samples were electrophoresed andtransferred to a Hybond-N nylon membrane by the capillary blottingmethod. The filters were washed at high stringency resulting in positiveclones with inserts ranging from 900 bp to 1700 bp.

Strongly hybridizing clones were analyzed by sequencing the ends of thecDNAs with forward and reverse pYES primers.

Analysis of the sequence data showed that some of these clones weretruncated cDNAs whereas others appeared to be full-length clones. Thenucleotide sequence and in FIG. 1) contains a cDNA insert of 1.4 kb.

Example 4

Expression of the Aminopeptidase in Aspergillus oryzae

To obtain high level production of aminopeptidase in Aspergillus oryzae,the cDNA insert from the pC1EXP3 clone was sub-cloned into pHD423 andco-transformed with the AmdS⁺ plasmid into Aspergillus oryzae asdescribed above.

The 1.4 kb cDNA insert from pC1EXP3 was isolated from pYES 2.0 byHindIII and Not I digestion, ligated to a Not I/HindIII cleaved pHD 423vector, and transformed into E. coli.

The resulting transformants were purified twice (see above) and assayedfor aminopeptidase activity as described above.

Transformant pA3EXP3/1 showed detectable aminopeptidase activity.

Example 5

Expression Level of Aminopeptidase Producing Transformant (pA3EXP3/1)

The amount and purity (level of expression) of secreted aminopeptidasefrom the transformant (pA3EXP3/1) was determined semi-quantitatively bySDS-PAGE using the non-transformed A. oryzae strain A01560 as a negativecontrol and the A. oryzae A01568 strain as positive control (see FIG.1).

A 36-37 kDa polypeptide could be seen in pA3EXP3/1, not present in thenegative control. The size of the recombinant aminopeptidase isapproximately 2 kDa higher than that of the native aminopeptidase (adouble band of about 35 kDa), possibly due to additional glycosylationor other types of post-translational modifications.

Example 6

Mass spectrometry showed that the recombinant aminopeptidase isglycosylated as the mass determined exceed the mass of the polypeptidecalculated from the cDNA sequence to be 32.4 kDa. The average mass ofthe recombinant aminopeptidase is 34.1 kDa with the masses ranging from33 kDa to 35 kDa.

The apparent molecular weight (M_(w)) determined by SDS-PAGE (asdescribed in "Materials and Methods" was found to be about 35 kDa.

Example 7

Fermentation of 35 kDa Aminopeptidase Producing Transformant

The Aspergillus oryzae transformant pA3EXP3/1 was grown in one litershake flasks containing 150 ml DAP 2C (pH=5.9) for three days at 30° C.

The amount of secreted aminopeptidase was estimated by SDS-PAGE analysisto approximately 0.5 g/liter supernatant.

Example 8

Expression of 35 kDa Aminopeptidase Clones in S. pombe

The full-length 35 kDa aminopeptidase cDNA clone pC1EXP3 wasre-transformed into Schizosaccharomyces pombe by electroporation. The1.4 kb cDNA insert was released from the pYES 2.0 vector by SpeI andNotI digestion and subcloned into the SpeI/NotI cleaved yeast expressionvector pP1, which is an E. coli/S. pombe shuttle vector containing theADH promoter, and assayed for aminopeptidase activity.

It was shown that one S. pombe transformant had strong aminopeptidaseactivity, indicating that S. pombe is able to synthesize and secrete afunctionally active 35 kDa aminopeptidase from Aspergillus oryzaeA01568.

Example 9

Organization and Expression of the Aminopeptidase Gene

The copy number of the aminopeptidase gene in the A. oryzae A01568genome was determined by Southern blot hybridization described in the"Materials and Methods" section. Total DNA isolated from A. oryzae wasdigested to completion with BamHI, BglII, EcoRI or HindIII andhybridized with the aminopeptidase cDNA. The aminopeptidase probedetects only single strongly hybridizing fragments in each case exceptin BamHI digestion, which gives two hybridizing fragments due to a BamHIsite at nucleotide position 640. This indicates that the aminopeptidasegene is present as a single copy in the A. oryzae A01568 genome.

Example 10

To study the expression of the 35 kDa aminopeptidase gene, poly (A)⁺ RNAextracted from A. oryzae A01568 mycelium was subjected to Northern blotanalysis. Probing of the blotted RNA with the aminopeptidase cDNArevealed two species of mRNA of approximately 1.45 and 1.55 kilobases.These two mRNAs do not appear to represent transcripts of two distinctgenes since the same pattern of hybridization was observed when thefilters were washed at high stringency. The difference in size of thetwo mRNAs could be due to different lengths of 3' untranslated region,because of two polyadenylation sites, in accordance with the two cDNAspecies isolated from the Aspergillus oryzae cDNA library, onecorresponding to clones pC1EXP3 and another one corresponding topC1EXP4, which is 120 bp shorter. Both mRNAs encode the sameaminopeptidase.

Example 11

Removing Bitter Taste from Whey Protein Hydrolysate

The fermentation broth of Aspergillus oryzae transformant pA3EXP3/1 wastested for the ability to debitter a solution containing 8% (w/w)protease hydrolysed whey protein.

The aminopeptidase activity of fermentation broth was 5.58 LAPU/g.

The substrate protein solution was tested in flasks containing 100 g ofthe 8% protein hydrolysate. The fermentation broth exhibitingaminopeptidase activity was added until the relationship between enzymeand substrate (E/S) was 0.25%, calculated on the basis of a product with5000 LAPU/g (equivalent to 12.5 LAPU/g protein), and was thenre-hydrolysed for 6 hours at 50° C., pH 7.0.

The pH and the osmolarity was determined (using standard methods) after1 minute and 6 hours, respectively. % DH was determined using theTNSB-method (described above) and % FAA (% free amino acids) wasdetermined using standard methods.

    ______________________________________                                                                          % DH                                           pH after pH after Increase by TNBS- total %                                  Aminopeptidase 1 minute 6 hours mOsm method FAA                             ______________________________________                                        Transformant                                                                             6.47     6.65    59    7.01*  13                                     Blind 6.61 6.81  1   3                                                      ______________________________________                                         *the increase of DH caused by the hydrolysis of the substrate by the          aminopeptidase. DH of the blind is 28%                                   

A sample of debittered hydrolysate containing 13% FFA was analyzed forthe content of leucine. It was found that leucine constituted about2.7%. while leucine constituted about 0.3% of the blind.

Example 12

Taste Test

The taste of debittered protein hydrolysate was assessed by a tastingpanel of 5 persons using a Bitterness Index (BI) between 0 (not bitter)and 10 (blind).

The bitterness, of a sample of bitter tasting 3.5% protein hydrolysate(blind) and a the transformant, was assessed.

The Bitterness Index (BI) for the protein hydrolysate sample subjectedto the transformant was found to be in the range of about 6.2.

This result shows that the 35 kDa aminopeptidase had debittered theprotein hydrolysate samples.

Example 13

Bread Flavour, Dough Stickiness and Crumb Structure

The flavour, dough stickiness and crumb structure of bread preparedusing from 0 to 300 LAPU per kg flour were compared with bread preparedusing the commercial product Flavourzyme®. The bread were prepared andassessed as described above in the "Material and Methods" section.

The following result (average of duplication) were found:

    ______________________________________                                                           Avg.                                                          LAPU/kg vol.  Dough Crumb                                                    Enzyme(s) flour Index Flavour stickiness structure                          ______________________________________                                        Flavourzyme ®                                                                       80       112    4     3      crumby and                                    open                                                                     Enzyme  0 100 3 4 as control                                                  of 10 100 3 4 as control                                                      the 30 100 3 4 as control                                                     Invention 50 100 3.25 4 as control                                             80  98 3.5 4 as control                                                       100  102 3.5 4 as control                                                     150   97 4 4 as control                                                       200   99 4 4 as control                                                       300  100 4 4 as control                                                    ______________________________________                                    

As can be seen from the above table the 35 kDa aminopeptidase of theinvention gives a significant flavour enhancement in the form of a"fresh baked" bread smell when added in amounts from 30 to 300 LAPU perkg flour. The crumb structure is not affected by the aminopeptidase ofthe invention. The dough stickiness is improved in comparison to thecommercial protease/peptidase complex Flavourzyme®.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof. Accordingly, the scope of the invention is to beconstrued in accordance with the substance defined by the claims below.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 15                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1409 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (B) STRAIN: Aspergillus - #oryzae A01568                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION:52..1183                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - CGAGCATTCC GTTCGTATCG ACTTGGTGGT ACACGCTTTC GTTCTCTCAA G - #ATG        CGT      57                                                                                       - #                  - #                  - #   Met       Arg                                                                                               - #                  - #                  - #     1         - - TTC CTC CCC TGC ATC GCG ACT TTG GCA GCC AC - #G GCC TCT GCC CTT GCT          105                                                                       Phe Leu Pro Cys Ile Ala Thr Leu Ala Ala Th - #r Ala Ser Ala Leu Ala                     5        - #          10        - #          15                      - - ATT GGA GAC CAT GTA CGC TCG GAC GAT CAG TA - #T GTC CTA GAA CTC GCC          153                                                                       Ile Gly Asp His Val Arg Ser Asp Asp Gln Ty - #r Val Leu Glu Leu Ala                20             - #     25             - #     30                          - - CCG GGA CAA ACG AAA GTT GTG ACG GAA GCA GA - #G AAA TGG GCT CTG AGA          201                                                                       Pro Gly Gln Thr Lys Val Val Thr Glu Ala Gl - #u Lys Trp Ala Leu Arg            35                 - # 40                 - # 45                 - # 50       - - GCT GAG GGC AAG CGT TTC TTC GAT ATA ACC GA - #A CGG GCC AGT AGC CTG          249                                                                       Ala Glu Gly Lys Arg Phe Phe Asp Ile Thr Gl - #u Arg Ala Ser Ser Leu                            55 - #                 60 - #                 65              - - GAA CTC GCA TCG AAC AAG AAA CAA AAG CTC GC - #G GTT ACC TAC CCC GAT          297                                                                       Glu Leu Ala Ser Asn Lys Lys Gln Lys Leu Al - #a Val Thr Tyr Pro Asp                        70     - #             75     - #             80                  - - TCC GTG CAA CAC AAC GAG ACC GTG CAA AAT CT - #G ATC AAG TCG CTC GAC          345                                                                       Ser Val Gln His Asn Glu Thr Val Gln Asn Le - #u Ile Lys Ser Leu Asp                    85         - #         90         - #         95                      - - AAA AAG AAC TTC GAA ACC GTT CTC CAG CCG TT - #C TCG GAG TTC CAC AAT          393                                                                       Lys Lys Asn Phe Glu Thr Val Leu Gln Pro Ph - #e Ser Glu Phe His Asn               100              - #   105              - #   110                          - - CGC TAT TAC AAG AGC GAC AAT GGC AAG AAA TC - #A TCC GAG TGG CTG CAA          441                                                                       Arg Tyr Tyr Lys Ser Asp Asn Gly Lys Lys Se - #r Ser Glu Trp Leu Gln           115                 1 - #20                 1 - #25                 1 -      #30                                                                              - - GGC AAG ATT CAG GAA ATC ATC TCC GCC AGT GG - #A GCA AAG GGA GTC        ACT      489                                                                    Gly Lys Ile Gln Glu Ile Ile Ser Ala Ser Gl - #y Ala Lys Gly Val Thr                          135  - #               140  - #               145              - - GTG GAG CCT TTC AAA CAC TCC TTC CCG CAG TC - #G AGT CTG ATT GCG AAA          537                                                                       Val Glu Pro Phe Lys His Ser Phe Pro Gln Se - #r Ser Leu Ile Ala Lys                       150      - #           155      - #           160                  - - ATC CCC GGC AAG AGT GAC AAG ACC ATC GTG CT - #T GGA GCG CAT CAG GAC          585                                                                       Ile Pro Gly Lys Ser Asp Lys Thr Ile Val Le - #u Gly Ala His Gln Asp                   165          - #       170          - #       175                      - - TCC ATC AAC CTT GAT TCA CCC TCA GAG GGC CG - #T GCA CCG GGA GCT GAT          633                                                                       Ser Ile Asn Leu Asp Ser Pro Ser Glu Gly Ar - #g Ala Pro Gly Ala Asp               180              - #   185              - #   190                          - - GAC GAT GGA TCC GGC GTT GTT ACC ATT CTC GA - #A GCC TTC CGC GTT CTC          681                                                                       Asp Asp Gly Ser Gly Val Val Thr Ile Leu Gl - #u Ala Phe Arg Val Leu           195                 2 - #00                 2 - #05                 2 -      #10                                                                              - - CTG ACG GAC GAG AAG GTC GCA GCC GGT GAG GC - #T CCG AAC ACC GTT        GAG      729                                                                    Leu Thr Asp Glu Lys Val Ala Ala Gly Glu Al - #a Pro Asn Thr Val Glu                          215  - #               220  - #               225              - - TTC CAC TTC TAT GCC GGA GAG GAG GGT GGT CT - #G CTG GGA AGT CAG GAC          777                                                                       Phe His Phe Tyr Ala Gly Glu Glu Gly Gly Le - #u Leu Gly Ser Gln Asp                       230      - #           235      - #           240                  - - ATC TTC GAG CAG TAC TCG CAG AAA AGC CGA GA - #C GTG AAA GCC ATG CTT          825                                                                       Ile Phe Glu Gln Tyr Ser Gln Lys Ser Arg As - #p Val Lys Ala Met Leu                   245          - #       250          - #       255                      - - CAA CAG GAT ATG ACG GGT TAT ACT AAA GGC AC - #A ACC GAT GCT GGA AAG          873                                                                       Gln Gln Asp Met Thr Gly Tyr Thr Lys Gly Th - #r Thr Asp Ala Gly Lys               260              - #   265              - #   270                          - - CCG GAG TCG ATC GGT ATC ATC ACT GAC AAT GT - #C GAT GAG AAC CTG ACC          921                                                                       Pro Glu Ser Ile Gly Ile Ile Thr Asp Asn Va - #l Asp Glu Asn Leu Thr           275                 2 - #80                 2 - #85                 2 -      #90                                                                              - - AAG TTC CTG AAG GTC ATT GTC GAT GCT TAT TG - #C ACT ATC CCG ACC        GTC      969                                                                    Lys Phe Leu Lys Val Ile Val Asp Ala Tyr Cy - #s Thr Ile Pro Thr Val                          295  - #               300  - #               305              - - GAT TCG AAA TGC GGA TAC GGA TGC TCT GAC CA - #T GCT TCT GCC ACG AAG         1017                                                                       Asp Ser Lys Cys Gly Tyr Gly Cys Ser Asp Hi - #s Ala Ser Ala Thr Lys                       310      - #           315      - #           320                  - - TAT GGT TAT CCC GCC GCA TTC GCA TTC GAG TC - #A GCC TTT GGC GAC GAC         1065                                                                       Tyr Gly Tyr Pro Ala Ala Phe Ala Phe Glu Se - #r Ala Phe Gly Asp Asp                   325          - #       330          - #       335                      - - AGC CCT TAC ATT CAC TCG GCT GAT GAT ACG AT - #T GAG ACC GTC AAC TTT         1113                                                                       Ser Pro Tyr Ile His Ser Ala Asp Asp Thr Il - #e Glu Thr Val Asn Phe               340              - #   345              - #   350                          - - GAC CAT GTG CTG CAA CAC GGC AAA CTG ACT CT - #T GGA TTT GCA TAT GAG         1161                                                                       Asp His Val Leu Gln His Gly Lys Leu Thr Le - #u Gly Phe Ala Tyr Glu           355                 3 - #60                 3 - #65                 3 -      #70                                                                              - - CTT GCC TTC GCA GAT TCG CTG T AAGGCTTATG ACGA - #CGGTTG TATGAGCGAG          1213                                                                      Leu Ala Phe Ala Asp Ser Leu                                                                   375                                                            - - AGATCCAGTC CAACAGTGTG TATAATATGT GGGCCCGTGT TCAAATAGCA CT -             #TTGATTTA   1273                                                                 - - GCCAGTGAGT AGCTTTGGTG GCGAAAATGG AGGCCGAATT CTAGGCAACA TC -            #GAACTGGA   1333                                                                 - - GGCTGTCAGG GGCGCATCAC AAGAAGTTTT GAGCTACATA AGCGAGATAA AA -            #GTCAGAAA   1393                                                                 - - AAAAAAAAAA AAAAAA             - #                  - #                      - #  1409                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 377 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Arg Phe Leu Pro Cys Ile Ala Thr Leu Al - #a Ala Thr Ala Ser Ala        1               5 - #                 10 - #                 15              - - Leu Ala Ile Gly Asp His Val Arg Ser Asp As - #p Gln Tyr Val Leu Glu                   20     - #             25     - #             30                  - - Leu Ala Pro Gly Gln Thr Lys Val Val Thr Gl - #u Ala Glu Lys Trp Ala               35         - #         40         - #         45                      - - Leu Arg Ala Glu Gly Lys Arg Phe Phe Asp Il - #e Thr Glu Arg Ala Ser           50             - #     55             - #     60                          - - Ser Leu Glu Leu Ala Ser Asn Lys Lys Gln Ly - #s Leu Ala Val Thr Tyr       65                 - # 70                 - # 75                 - # 80       - - Pro Asp Ser Val Gln His Asn Glu Thr Val Gl - #n Asn Leu Ile Lys Ser                       85 - #                 90 - #                 95              - - Leu Asp Lys Lys Asn Phe Glu Thr Val Leu Gl - #n Pro Phe Ser Glu Phe                  100      - #           105      - #           110                  - - His Asn Arg Tyr Tyr Lys Ser Asp Asn Gly Ly - #s Lys Ser Ser Glu Trp              115          - #       120          - #       125                      - - Leu Gln Gly Lys Ile Gln Glu Ile Ile Ser Al - #a Ser Gly Ala Lys Gly          130              - #   135              - #   140                          - - Val Thr Val Glu Pro Phe Lys His Ser Phe Pr - #o Gln Ser Ser Leu Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Lys Ile Pro Gly Lys Ser Asp Lys Thr Il - #e Val Leu Gly Ala        His                                                                                             165  - #               170  - #               175             - - Gln Asp Ser Ile Asn Leu Asp Ser Pro Ser Gl - #u Gly Arg Ala Pro Gly                  180      - #           185      - #           190                  - - Ala Asp Asp Asp Gly Ser Gly Val Val Thr Il - #e Leu Glu Ala Phe Arg              195          - #       200          - #       205                      - - Val Leu Leu Thr Asp Glu Lys Val Ala Ala Gl - #y Glu Ala Pro Asn Thr          210              - #   215              - #   220                          - - Val Glu Phe His Phe Tyr Ala Gly Glu Glu Gl - #y Gly Leu Leu Gly Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Asp Ile Phe Glu Gln Tyr Ser Gln Lys Se - #r Arg Asp Val Lys        Ala                                                                                             245  - #               250  - #               255             - - Met Leu Gln Gln Asp Met Thr Gly Tyr Thr Ly - #s Gly Thr Thr Asp Ala                  260      - #           265      - #           270                  - - Gly Lys Pro Glu Ser Ile Gly Ile Ile Thr As - #p Asn Val Asp Glu Asn              275          - #       280          - #       285                      - - Leu Thr Lys Phe Leu Lys Val Ile Val Asp Al - #a Tyr Cys Thr Ile Pro          290              - #   295              - #   300                          - - Thr Val Asp Ser Lys Cys Gly Tyr Gly Cys Se - #r Asp His Ala Ser Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Lys Tyr Gly Tyr Pro Ala Ala Phe Ala Ph - #e Glu Ser Ala Phe        Gly                                                                                             325  - #               330  - #               335             - - Asp Asp Ser Pro Tyr Ile His Ser Ala Asp As - #p Thr Ile Glu Thr Val                  340      - #           345      - #           350                  - - Asn Phe Asp His Val Leu Gln His Gly Lys Le - #u Thr Leu Gly Phe Ala              355          - #       360          - #       365                      - - Tyr Glu Leu Ala Phe Ala Asp Ser Leu                                          370              - #   375                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Primer                                            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - TCNACRTTRT CNGTNATNAT YTCNAT          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Primer                                            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - GARACNGTNC ARAAYCTNAT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Primer                                            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - GAYAARAARA AYTTYGAWAC NGT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - Tyr Pro Asp Ser Val Gln His Xaa Glu Thr Va - #l Gln Asn Leu Ile Lys      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - Gly Val Thr Val Glu Pro Phe Lys                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - Val Ile Val Asp Ala Tyr Cys Thr Ile Pro Th - #r Val Asp Ser Lys          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - Gly Thr Thr Asp Ala Gly Lys Pro Glu Ser Il - #e Glu Ile Ile Thr Asp      1               5   - #                10  - #                15               - - Asn Val Asp Glu Asn Leu Thr Lys                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - Tyr Pro Asp Ser Val Gln His Xaa Glu Thr Va - #l Gln Asn Leu Ile Lys      1               5   - #                10  - #                15               - - Ser Leu Asp Lys Lys Asn Phe Glu Thr Val Le - #u Gln Pro                              20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - Asn Phe Glu Thr Val Leu Gln Pro Phe Ser Gl - #u Phe His Asn Arg Tyr     Tyr Lys                                                                        1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1272 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (B) STRAIN: Aspergillus - #oryzae A01568                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - CTCGAGCATT CCGTTCGTAT CGACTTGGTG GTACACGCTT TCGTTCTCTC AA -             #GATGCGTT     60                                                                 - - TCCTCCCCTG CATCGCGACT TTGGCAGCCA CGGCCTCTGC CCTTGCTATT GG -            #AGACCATG    120                                                                 - - TACGCTCGGA CGATCAGTAT GTCCTAGAAC TCGCCCCGGG ACAAACGAAA GT -            #TGTGACGG    180                                                                 - - AAGCAGAGAA ATGGGCTCTG AGAGCTGAGG GCAAGCGTTT CTTCGATATA AC -            #CGAACGGG    240                                                                 - - CCAGTAGCCT GGAACTCGCA TCGAACAAGA AACAAAAGCT CGCGGTTACC TA -            #CCCCGATT    300                                                                 - - CCGTGCAACA CAACGAGACC GTGCAAAATC TGATCAAGTC GCTCGACAAA AA -            #GAACTTCG    360                                                                 - - AAACCGTTCT CCAGCCGTTC TCGGAGTTCC ACAATCGCTA TTACAAGAGC GA -            #CAATGGCA    420                                                                 - - AGAAATCATC CGAGTGGCTG CAAGGCAAGA TTCAGGAAAT CATCTCCGCC AG -            #TGGAGCAA    480                                                                 - - AGGGAGTCAC TGTGGAGCCT TTCAAACACT CCTTCCCGCA GTCGAGTCTG AT -            #TGCGAAAA    540                                                                 - - TCCCCGGCAA GAGTGACAAG ACCATCGTGC TTGGAGCGCA TCAGGACTCC AT -            #CAACCTTG    600                                                                 - - ATTCACCCTC AGAGGGCCGT GCACCGGGAG CTGATGACGA TGGATCCGGC GT -            #TGTTACCA    660                                                                 - - TTCTCGAAGC CTTCCGCGTT CTCCTGACGG ACGAGAAGGT CGCAGCCGGT GA -            #GGCTCCGA    720                                                                 - - ACACCGTTGA GTTCCACTTC TATGCCGGAG AGGAGGGTGG TCTGCTGGGA AG -            #TCAGGACA    780                                                                 - - TCTTCGAGCA GTACTCGCAG AAAAGCCGAG ACGTGAAAGC CATGCTTCAA CA -            #GGATATGA    840                                                                 - - CGGGTTATAC TAAAGGCACA ACCGATGCTG GAAAGCCGGA GTCGATCGGT AT -            #CATCACTG    900                                                                 - - ACAATGTCGA TGAGAACCTG ACCAAGTTCC TGAAGGTCAT TGTCGATGCT TA -            #TTGCACTA    960                                                                 - - TCCCGACCGT CGATTCGAAA TGCGGATACG GATGCTCTGA CCATGCTTCT GC -            #CACGAAGT   1020                                                                 - - ATGGTTATCC CGCCGCATTC GCATTCGAGT CAGCCTTTGG CGACGACAGC CC -            #TTACATTC   1080                                                                 - - ACTCGGCTGA TGATACGATT GAGACCGTCA ACTTTGACCA TGTGCTGCAA CA -            #CGGCAAAC   1140                                                                 - - TGACTCTTGG ATTTGCATAT GAGCTTGCCT TCGCAGATTC GCTGTAAGGC TT -            #ATGACGAC   1200                                                                 - - GGTTGTATGA GCGAGAGATC CAGTCCAACA GTGTGTATAA TATGTGGGCC CG -            #TGTTCAAA   1260                                                                 - - TAGCACTTAA AA              - #                  - #                      - #     1272                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GARACNGTNC ARAAYCTNAT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GAYAARAARA AYTTYGANAC NGT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TCNACRTTRT CNGTNATNAT YTCNAT          - #                  - #                  26                                                                    __________________________________________________________________________

We claim:
 1. An isolated and purified enzyme exhibiting aminopeptidase activity comprising one or more of the amino acid sequences of SEQ ID NO:6, 7, 8, 9, 10, and 11, derived from a strain of Aspergillus oryzae.
 2. The enzyme of claim 1, where the enzyme comprises the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, or an enzymatically active fragment thereof.
 3. The enzyme of claim 1, where the enzyme comprises the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:
 11. 4. The enzyme of claim 1, wherein the enzyme comprises a molecular weight of about 35 kDa determined by SDS-PAGE.
 5. The enzyme of claim 1 obtained from E. coli, DSM
 9965. 6. An isolated and purified enzyme exhibiting aminopeptidase activity comprising the amino acid sequence of SEQ ID NO: 2, or an enzymatically active fragment thereof.
 7. The enzyme of claim 6, wherein the enzyme comprises a molecular weight of about 35 kDa determined by SDS-PAGE.
 8. The enzyme of claim 6, derivable from a strain of the genus Aspergillus.
 9. The enzyme of claim 8, derivable from a strain of Aspergillus oryzae.
 10. The enzyme of claim 6, obtained from E. coli, DSM
 9965. 11. An enzyme preparation comprising the enzyme of claim
 6. 12. A bread-improving or a dough-improving composition comprising the enzyme of claim
 6. 13. An enzyme preparation comprising the enzyme of claim
 1. 14. A bread-improving or a dough-improving composition comprising the enzyme of claim
 1. 